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Proc Natl Acad Sci U S A. Sep 26, 2006; 103(39): 14572–14577.
Published online Sep 14, 2006. doi:  10.1073/pnas.0606127103
PMCID: PMC1600001
Microbiology

This article has been retractedRetraction in: Proc Natl Acad Sci U S A. 2007 February 27; 104(9): 3668    See also: PMC Retraction Policy

The herpesvirus glycoproteins B and H·L are sequentially recruited to the receptor-bound gD to effect membrane fusion at virus entry

Abstract

Four glycoproteins (gD, gB, gH, and gL) are required for herpes simplex virus entry into the cell or for cell–cell fusion in transfected cells. gD serves as the receptor-binding glycoprotein and as the trigger of fusion; the other three execute fusion between the viral envelope and the plasma and endocytic membranes or the membranes of adjacent cells and are highly conserved among members of the herpesvirus family. Details of the interaction of gD with gB, gH, and gL were not known. Here, we report that the four glycoproteins assemble into a complex initiated by the interaction of gD with its cellular receptor. gB is recruited to the gD–receptor complex next, even in the absence of gH·gL. gH·gL is recruited next, but only to the receptor–gD–gB ensemble. A complex with the composition receptor–gD–gB–gH·gL is assembled transiently with a life span of 15–30 min in cells exposed to virus but can also be found in infected cells and in cells committed to form polykaryocytes after transfection of the glycoprotein quartet. The results indicate that the complex assembly is a critical step in the process of virus entry and fusion, and that no viral protein other than those that participate in the complex itself is required for complex assembly. These findings imply critical protein–protein interactions among the quartet as herpes simplex virions enter the cells and at cell–cell fusion, define a specific order of recruitment, and place gH·gL as the last link in the process of glycoprotein recruitment to the complex.

Keywords: complex, herpes simplex virus

The entry of herpes simplex virus (HSV) into cells takes place in three steps. In the first, two viral glycoproteins, gC and gB, bind to heparan sulfate proteoglycans (1). In the second step, another glycoprotein, gD, interacts with at least one of two alternative protein receptors: nectin 1, an intercellular adhesion molecule, or herpesvirus entry mediator (HVEM), a member of tumor necrosis factor α receptor family (25). In the third step, gH, gL, and gB execute the fusion of viral envelope with the cell membranes, either plasma or endosomal (refs. 511; for reviews, see refs.12 and 13). Some of the details of the last two steps have been elucidated recently. Thus, receptor-mediated activation of gD takes the form of a conformational change (1416). The gD ectodomain is organized in two distinct regions, an N terminus (amino acids 1–260) carrying the receptor binding sites, and the C terminus (amino acids 260–310) carrying the profusion domain required to trigger fusion but not for receptor binding (14). In the unliganded state, the virion gD adopts a conformation in which the flexible C terminus of the ectodomain folds back, wraps the N terminus, and masks receptor-binding sites. At receptor binding, the ectodomain C terminus is displaced from its binding site on the N terminus, and the receptor-binding sites are unmasked and become occupied by the receptor (15, 16). How exactly the displaced C terminus carrying the profusion domain triggers fusion remains to be elucidated.

The executors of fusion are glycoproteins B, H, and L (5, 6, 8, 9). It can be assumed that in the receptor-bound conformation gD recruits or activates the fusion executors, but the nature of the interaction between gD and the fusion executors is not known. Of the three executors, gH appears to contain elements associated with fusion of juxtaposed membranes, i.e., a hydrophobic α-helix 1 (residues 377–397) with properties typical of a fusion peptide loop and two heptad repeats (17). α-Helix 1 is positionally conserved in all of the gH orthologs across the herpesviridae family; in HSV-2 it is located in a loop made of two cysteins. α-Helix 1 is able to interact with biological membranes, can convert a soluble glycoprotein (gD amino acid residues 1–260) into a membrane-bound glycoprotein, and can be functionally replaced by fusion peptides derived from glycoproteins of other, unrelated viruses (17). A peptide with the sequence of α-helix 1 induces fusion of liposomes and exhibits a strong flexibility documented as ability to adopt an α-helical conformation (18, 19). These properties strongly argue in favor of α-helix 1 as a candidate fusion peptide loop. Two heptad repeats, capable of forming coiled-coils and interacting with each other, form a structure of increased α-helical content and are potentially suitable to form a six-helix bundle (2022). Additional elements in gH are a second predicted α-helical domain of lower hydrophobicity than the candidate fusion peptide (amino acids 513–531) and a pretransmembrane sequence (amino acids 626–644) with predicted propensity to partition at membrane interface (18, 19).

gL is a soluble glycoprotein that forms a heterodimer with gH and is required for gH to adopt its correct conformation and to be transported to the plasma membrane (23).

gB binds heparan sulfate glycosaminoglycans (1). Its crystal structure reveals a trimer with a coiled-coil core that relates gB to viral fusion glycoproteins, even though a canonical fusion peptide has not been detected by biochemical, molecular, or structural analyses. A putative fusion loop appears suboptimal for membrane insertion (24). gB orthologs exhibit receptor-binding activity (25, 26). The respective roles and mechanisms played by gB and gH·gL in fusion execution are unknown.

The nature of the interactions between the complex formed by gD plus its receptor and the executors of fusion is critical in understanding the mechanisms by which HSV (and by extension all other herpesviruses) enters cells. In other herpesviruses, a few well defined complexes, mainly involving gH·gL, have been reported, although they were not detected at virus entry, but in virions or in infected cells, and their functional significance is unclear (2731). Efforts to reveal complexes among HSV glycoproteins by cross-linking experiments revealed high-molecular-mass heterooligomers, detectable even when infections were carried out with viral mutants lacking gB, gH, or gL (32, 33). More recently, Fuller and coworkers (34) reported that HSV-infected HEp-2 cells harbor a HVEM–gD–gH complex. The presence of gB and gL in the complex was not investigated.

In this article, we show that in receptor-positive cells infected with herpes simplex virions or in cells transfected with the glycoprotein quartet, the fusion executors interact with the receptor-bound gD in an orderly sequence. The binding of gB to gD requires the presence of nectin 1 or HVEM, and is followed by the recruitment of gH·gL. In infected cells, a fraction of the glycoproteins can assemble with receptor-bound gD into a complex made of the same constituents as the one seen at virus entry.

Results

gB Participates in a Complex Made of HVEM, gD, and gH·gL.

The ultimate objective of this work was to analyze the assembly of glycoprotein complex at HSV entry and in infected and transfected cells. Because our study was prompted by the studies reported by Fuller and coworkers (34), the initial experiments were carried out with cells rather than with virions. The first question we addressed was whether gB and gL participate in a complex made of HVEM–gD and gH. HEp-2 cells infected with HSV-1(F) were lysed, gD was immunoprecipitated with three different Abs to gD, and the precipitate was immunoblotted to identify the presence of coprecipitated viral glycoproteins. Fig. 1A and B shows that, of the three mAbs to gD (R8, R45, and ZC15), only R8 coimmunoprecipitated gH, gB, and gL, as visualized by immunoblotting of electrophoretically separated proteins. Next, we showed that anti-HVEM antibody also coimmunoprecipitated the four glycoproteins (Fig. 1). As expected, the complex was specifically detected in the infected cells. An irrelevant Ab did not coimmunoprecipitate any one of the four glycoproteins (data not shown). As a specificity control, gC, an abundant glycoprotein involved in HSV attachment to cells, failed to be recruited to the complex (Fig. 1B). The results confirm and extend a previous report (34) and show that infected cells harbor a complex that includes HVEM–gD–gB–gH·gL. We verified that the receptor–glycoprotein complex was detectable also in another cell line. Fig. 1B shows that also in infected 293 cells, polyclonal Ab (pAb) R8 to gD or R140 to HVEM coimmunoprecipitated gB, gH, and gL. Thus, infected cells contain a multiprotein complex that can be coimmunoprecipitated with Abs to either gD or HVEM, whose composition is HVEM, gD, gB, and gH·gL.

Fig. 1.
Electrophoretic and WB analysis of proteins coimmunoprecipitated by Abs to gD or to HVEM from lysates of HSV-1(F)-infected HEp-2 or 293 cells. gD or HVEM were immunoprecipitated (IP) with the Abs indicated in parentheses from lysates of HSV-1(F)-infected ...

Comparison of the amounts of gD immunoprecipitated by R8 Ab to gD, or coimmunoprecipitated R140 Ab to HVEM, clearly indicated that the amounts of gD coimmunoprecipitated by R140 were smaller than that directly immunoprecipitated by anti-gD Ab. Conversely, gD was present in the complex-unbound fractions in large quantity when immunoprecipitation was carried out with HVEM Ab (Fig. 1B) and in small quantity when immunoprecipitation was carried with gD Ab (data not shown). This difference suggested that the fraction of gD recruited to the complex was only a minor fraction of the total amount of gD present in the cells (Fig. 1B). The amounts of gB, gH, and gL coimmunoprecipitated by Ab to gD or to HVEM did not markedly differ (Fig. 1 A and B). However, even for gB and gH·gL, Western blot (WB) analysis of the fractions unbound to the complex showed that it contained large amounts of the three glycoproteins (Fig. 1B). Similar results were obtained both in HEp-2 and in 293 cells (Fig. 1 A and B). We interpret these results as evidence that the amounts of gD, gB, gH, and gL recruited to the complex are but a minor fraction of the total amounts of glycoproteins present in the infected cells.

The Glycoprotein Quartet Assembles into a Complex also with Nectin 1.

To investigate whether the glycoprotein quartet assembles into a complex also with nectin 1, the receptor was immunoprecipitated from infected 293 cells with three mAbs (CK4, CK9, and CK35) (35); the presence of gD and gB in the immunocomplex fraction was considered diagnostic of complex formation. As shown in Fig. 2, nectin 1 was capable of coimmunoprecipitating gD and gB.

Fig. 2.
WB analysis of proteins coimmunoprecipitated by Abs to HVEM or to nectin 1. HVEM or nectin 1 (Nec1) were immunoprecipitated (IP) with Abs indicated in parentheses from HSV-1(F)-infected 293 cells. Coimmunoprecipitated proteins were separated by using ...

The Glycoprotein Quartet Assembles into a Complex at HSV Entry.

To verify whether the glycoprotein quartet assembles into a complex at HSV entry, partially purified extracellular virions (50 pfu per cell) were allowed to absorb to cells at 4°C for 120 min, and the virus inoculum was removed. The cultures were then rinsed twice (0 time) and shifted to 37°C in a water bath (synchronization of virus entry) for 15, 30, or 45 min. gD or HVEM were immunoprecipitated from virus-cell lysates, and the coimmunoprecipitated proteins were identified by WB. Fig. 3A shows the results obtained with HVEM Ab; the results with R8 Ab to gD were essentially the same (data not shown). At 0 time (end of virus absorption), a small amount of gD was immunoprecipitated, likely because of virions absorbed to cells, whose gD was interacting with HVEM. gB, gH, and gL were not coimmunoprecipitated. At 15 min after shift to 37°C, but not at 30 or 45 min, gD, gB, gH, and gL were coimmunoprecipitated by HVEM Ab. Importantly, the complex was specifically formed at virus entry, because no gB, gH, or gL were coimmunoprecipitated when cells were exposed to virions lacking gD (gD−/− virions) or when WT virions were absorbed to receptor-negative CHO cells (Fig. 3). The results highlight the formation of a complex made of HVEM and the quartet, transiently at virus entry. The kinetics of complex formation is very rapid, about 15 min, and the lifespan of the complex is short, between 15 and 30 min. Although no complex other than that of gD and its receptor was detected in earlier works, these results are in accordance with a number of previous findings, especially with the notion that fusion of herpes simplex virions with cells takes place a few minutes after virus attachment, that virion gD formed a complex with nectin 1 within minutes after virus exposure to cells, that nectin 1 was not removed from the cell surface but was modified during the first 30 min of HSV infection, and that this was followed by relocation of nectin 1 from cell–cell contact areas between 1 and 3 h postinfection (10, 3638).

Fig. 3.
WB analysis of proteins coimmunoprecipitated by Abs to HVEM or gD from cells incubated with virions. The 293 or CHO cells were incubated with HSV-1(F) virions (wt) (50 pfu per cell) or gD−/− virions (50 pfu-equivalents per cell) for 120 ...

A Glycoprotein Complex Is Assembled in Cells Transfected with the Quartet.

Pivotal to determining whether viral proteins other than the quartet are required for complex assembly is the ability to reconstitute the complex in transfected cells. Previously, it was documented that transfection of receptor-negative cells with gD and its receptor results in a receptor–gD complex present in plasma and intracellular membranes (39). In a first experiment, 293 cells were transfected with plasmids encoding the glycoprotein quartet plus a 2-fold amount of HVEM plasmid. The highest amounts of coimmunoprecipitated gB, as well as of gH and gL, were found at 24 h posttransfection (p.t.) (Fig. 4), i.e., before polykaryocyte formation; no gB (or gH and gL) was coimmunoprecipitated at 36 h p.t., i.e., at the time when cells had already formed polykaryocytes (Fig. 4). Immunoprecipitation with an unrelated Ab did not pull down any protein. As observed in infected cells (Fig. 1B), the amount of gD coimmunoprecipitated by HVEM Ab was smaller than that directly immunoprecipitated by anti-gD Ab (Fig. 4; compare gD bands in the two series of immunoprecipitations), and furthermore, only a portion of gB was in complex with HVEM or gD, the majority being unbound to the complex (data not shown). The conclusions are threefold: (i) under conditions in which cells are committed to fuse with each other, the glycoprotein quartet assembles into a receptor- and gD-dependent complex; (ii) no viral protein other than the quartet is required for complex assembly; and (iii) only a minor fraction of the glycoproteins assemble into the complex, and the majority remain unengaged.

Fig. 4.
Time course of glycoprotein complex formation in transfected cells. 293 cells were transfected with expression plasmids for gD, gB, and gH·gL and twice the amount of HVEM plasmid. At the indicated hour p.t., cells were harvested and HVEM or gD ...

Complex Assembly Strictly Depends on a gD Receptor.

The possibility to detect complex formation in transfected cells offered a chance to ask whether complex assembly depended on one of the gD receptors. The receptor-negative CHO cells were transfected with the quartet plus nectin 1, HVEM, or no receptor. gD was precipitated with Ab R8. The presence of coimmunoprecipitated gB was considered as diagnostic of complex formation. The results in Fig. 5 show that gB failed to be coimmunoprecipitated when neither receptor was present but was coimmunoprecipitated when either HVEM or nectin 1 was present.

Fig. 5.
Glycoprotein complex assembly requires a gD receptor. The receptor-negative CHO cells were transfected with expression plasmids for gD, gB, and gH·gL with HVEM (+HVEM), with nectin 1 (+Nec), or with no receptor (+No R). gD was immunoprecipitated ...

Requirements for Complex Assembly in Infected and Transfected Cells.

We analyzed the requirements for complex assembly in Hep-2 and 293 cells infected with deletion mutant viruses, and, in parallel, in 293 cells transfected with HVEM plus the quartet, or with glycoprotein mixtures in which one of the glycoproteins was omitted. The rationale was that, if complex formation takes place through a sequential order of recruitment, infection with a specific deletion mutant should lead to a partial complex lacking the deleted glycoprotein and the glycoproteins recruited downstream of it. Similarly, in cells transfected with mixtures lacking a given glycoprotein, the glycoprotein(s) recruited downstream of it should be missing from the complex. The coimmunoprecipitations gave the following results.

  • In ΔgD (gD−/+) (Δ = deletion) virus-infected cells, neither gB nor gH·gL were coimmunoprecipitated (Fig. 6 A and B), as previously reported for gB (34). Accordingly, in cells transfected with the glycoprotein mixture lacking gD [BHL (gB, gH, gL)], neither gB nor gH·gL were coimmunoprecipitated (Fig. 6C). In both infected and transfected cells, the results were the same irrespective of whether the immunoprecipitations were carried out with Ab R8 to gD or R140 to HVEM (results shown for gD).
    Fig. 6.
    Analysis of immuno- and coimmunoprecipitated proteins from cells infected with deletion viruses or transfected with the indicated mixtures. (A) HEp-2 cells. (BD) 293 cells. (A and B) Cells infected with WT virions (wt) or ΔgD, ΔgB, ...
  • In cells infected with the ΔgH (gH−/+) or ΔgL (gH−/+) viruses, gD coimmunoprecipitated gB (Fig. 6 A and B). Similarly, when gH or gL were omitted from the transfection mixture (DBL or DBH, respectively), gB was still coimmunoprecipitated by gD Ab (Fig. 6C).
  • In cells infected with the ΔgB (gB−/+) virus, gD failed to coimmunoprecipitate gH·gL. Similarly, when gB was omitted from the transfection mixture [DHL (gD, gH, gL)], gH and gL were not coimmunoprecipitated (Fig. 6C).
  • We verified whether an abundant glycoprotein involved in HSV attachment to cells, gC, was recruited to the complex. The results in Fig. 6B indicate that gC was not coimmunoprecipitated by gD Ab. Inclusion of gC in the transfection mixture [DBHLC (gD, gB, gH, gL, gC)] led to a complex that included the viral glycoproteins D, B, and H·L, but not gC, reinforcing evidence for the specificity of the complex (Fig. 6 C and D).
  • Attempts to verify the behavior of gL in ΔgH virus-infected cells, or in cells transfected with a gH-less mixture, provided unclear results. In some experiments, gL appeared to associate to gB (Fig. 6C). These results are of difficult interpretation because, under these conditions, gL is soluble, not associated to the membranes by means of gH, rapidly excreted from the cell, and not properly folded. Similarly, in cells infected with the ΔgL virus, gD coimmunoprecipitated gB, but not gH. We cannot distinguish whether the latter effect was the consequence of gH misfolding due to gL absence, of gH retention in endoplasmic reticulum, or of the fact that gL represents the actual molecule bound by gB. Altogether, whether gB interacts with the gH·gL heterodimer via gH or via gL remains to be verified with different experimental approaches.
  • In all instances, analysis of the lysates of infected and transfected cells verified the actual presence of the glycoproteins, according to the genotype of the viral mutant or according to the transfections (data shown only in Fig. 6D for the transfected cells). As illustrated in Fig. 1 for cells infected with WT virus, also in cells infected with deletion viruses or in transfected cells, the amounts of glycoproteins recruited to the complex was but a minor fraction of the total (data not shown).

Altogether, results with infected and transfected cells concordantly indicate that gB formed a complex with gD also in the absence of gH·gL, whereas gH·gL formed a complex with gD only when gB was present. The results imply that the sequential order of glycoprotein recruitment to the complex is receptor–gD–gB–gH·gL.

Discussion

Earlier studies have established that the interaction of gD with one of its receptors is an essential first step in the entry of HSV into infected cells, that the interaction with the receptor alters the conformation of gD such that gD activates the fusion executor glycoproteins gB, gH, and gL (24, 7, 1216). A central, unresolved question regarding the mechanisms by which HSV gains entry into cells is the interactions that take place among the quartet and the nature of the complexes formed by viral glycoproteins at the moment of fusion of the viral envelope with the cell membrane. In this article, we have shown the following.

  • Upon entry of HSV into cells, gD, gB, and gH·gL assembled into a complex that included a gD receptor. A complex containing this quartet of glycoproteins was assembled also in infected cells or in cells transfected with the four glycoproteins. The complex could be immunoprecipitated by Abs to gD, nectin 1, or HVEM. In infected and transfected cells, the quantities of the glycoproteins gD, gB, or gH found in the complex constituted only a fraction of the total.
  • The complex was not detected upon exposure of receptor-negative cells to virus, or in receptor-negative cells transfected with the quartet. It was not assembled in the absence of gD.
  • gB was recruited to the complex made of receptor plus gD also in cells infected with ΔgH or ΔgL viruses, or in cells transfected with gH- or gL-less mixtures. By contrast, in the absence of gB, neither gH nor gL were recruited to complex made of receptor plus gD.
  • The viral glycoproteins that formed the complex were gD and the three fusion executor glycoproteins. The complex did not include the abundant viral gC. The absence of gC from this complex is highly significant because gC is involved in the initial attachment to heparan sulfate proteoglycans.
  • The complex did not form in cells exposed to virions at 4°C. The complex was detected after exposure of cells to virus at 37°C, and its lifespan did not exceed 15 min, consistent with several lines of evidence indicating that the fusion of the envelope with the cell membrane occurs very rapidly after initial attachment (10, 36).

The significance of our results is as follows:

  • The quartet of glycoproteins essential for HSV entry and fusion assemble into a complex at virus entry and in infected cells. The gD receptor that drives complex formation may be either nectin 1 or HVEM. The same complex is assembled also in cells transfected with the quartet, implying that no additional viral protein other than those that participate in the complex itself is required. It follows that the quartet of glycoproteins essential for virus entry and cell–cell fusion are the viral components both necessary and sufficient for complex assembly, and that the complexes detectable at virus entry, in infected or transfected cells, contained the same viral constituents. The findings that the complex was assembled at virus entry and in transfected cells committed to form polykaryocytes, and failed to be assembled in the absence of either a receptor to gD or of gD, argue that complex assembly is a critical step in the process of virus entry and fusion.
  • The results show that the viral glycoproteins are recruited in a specific order. The receptor–gD complex is formed first and is followed by the binding of gB and then of gH·gL. As outlined in the Introduction, gH exhibits features typical of viral fusion glycoproteins and is a likely candidate executor of fusion (17, 20, 21). The finding that, with respect to the order of recruitment, gB occupies an intermediate position between gD and gH·gL leaves unanswered the question as to whether gB enables the recruitment, and possibly the refolding, of gH·gL, such that gH·gL execute fusion, or whether gB cooperates with gH·gL in fusion execution.
  • The finding of a small amount of receptor–gD–gB–gH·gL complexes in cells harvested late in infection with wild-type virus comes as no surprise. Two sets of findings bear on this issue. First, in this article, we show that the lifespan of this complex on entry of virions into cells is relatively short (≈15 min). Second, there is considerable evidence that expression of gD causes a reduced availability of receptor in transfected cells (39), a reflection of a key function of gD that prevents reentry of newly released progeny viruses into the cells from which they have emerged (40, 41). Consonant with these observations, Krummenacher et al. (37) reported down-modulation of nectin1 from the surface of infected cells. Moreover, they reported that in the first 1 h of infection, nectin 1 is modified such that it no longer reacts with an Ab directed to the gD-binding site on nectin 1, even though the total amount of nectin 1 on cell surface remains unchanged (38). In addition, cells cotransfected with nectin 1 and gD fail to bind a soluble form of gD, in contrast to cells expressing nectin 1 alone (39). Taken together, these three sets of observations argue that, in infected cells, gD actively engages receptor proteins into complexes and that in the complex the receptor undergoes a modification that precludes its further interaction with gD. It could be expected that the scavenging activity of gD would continue throughout the infectious process with the result that small amounts of receptor–gD–gB–gH·gL complexes can be detected even late in infection.
  • The sequentially ordered glycoprotein recruitment to the complex refines the current model of HSV entry. The receptor-mediated mechanism of gD activation involves a major switch in gD conformation that entails the displacement of the C terminus of gD from its binding site on the N terminus and the consequent exposure of the pro-fusion domain. The binary complex made of receptor and gD in this conformation creates a surface suitable for gB recruitment. With the involvement or not of a gB receptor, the recruited gB likely undergoes a conformational change that leads to recruitment of gH·gL and possibly to conformational changes to gH·gL such that gB–gH·gL adopt the fusion-active conformation and execute fusion. Much remains to be verified of this model. The possibility to detect critical protein–protein interactions will undoubtedly contribute to better define the sequentially ordered and coordinate process of glycoprotein recruitment and activation at virus entry and cell–cell fusion.

Materials and Methods

Cells and Viruses.

Cells were grown in Dulbecco’s modified minimum essential medium containing 5–10% FCS. The receptor-negative CHO cells were grown in α-medium. HSV-1(F) is described in ref. 42. The ΔgD F-gDβ, ΔgB-KΔT, ΔgH SCgHZ, and ΔgL mutants were grown and titrated in the respective complementing cells to produce complemented (gD−/+, gB−/+, gH−/+, and gL−/+) virions and in Vero cells to produce phenotypically negative virions (gD−/−) (69). In all cases, extracellular virions pelletted through a sucrose cushion were used.

Antibodies.

mAbs H170 to gD, H1817 to gB, and H633 to gC were from the Goodwin Institute (Plantation, FL). pAb R8 to gD (43), pAb ZC15 to gD C-tail (44), pAb R95 and pAb R140 to HVEM (34, 45), mAb VII.62.15 to gL (46), H12 (47), and CK4, CK9, and CK35 (35) have been described.

Transfections.

Transfections of CHO and 293 cells were carried out by using Arrest-in (Celbio, Milan, Italy). Cultures in T25 flasks, at 50% or lower confluency for CHO cells and 2 × 106 per flask for 293, were transfected 16 h after seeding, with mixtures containing 3 μg of pBEC encoding HVEM (2) and 1.5 μg each of expression plasmids encoding gD, gB, gH, and gL, except as otherwise stated. The glycoprotein plasmids are described in ref. 48. Transfected cells were harvested at 18–24 h after transfection, before syncytia formation, and immediately processed for glycoprotein extraction.

Immunoprecipitations and Western Blots.

Cells were lysed without freezing in EA buffer (50 mM hydroxyethylpiperazine-ethanesulfonic acid/250 mM NaCl/0.1% Nonidet P-40) or in PBS* (PBS containing 1% Nonidet P-40 and 1% deoxycholate), containing protease inhibitors (0.1 mM Na-p-tosyl-l-lysine chloromethylketone/0.1 mM tolylsulfonyl phenylalanyl chloromethyl ketone). Immunoprecipitations were carried out with the indicated Abs from lysates of infected or transfected cells, or of cells exposed to virions, as described in ref. 34. The immunocomplexes were harvested with protein A-Sepharose (Sigma, Milan, Italy). Proteins separated by SDS/PAGE were transferred to a Hybond ECL Nitrocellulose membrane (Amersham Pharmacia, Milan, Italy), reacted with appropriate Abs and for ECL.

Acknowledgments

We thank our colleagues for their generosity and continuous supply of critical reagents, in particular, Drs. G. H. Cohen (University of Pennsylvania, Philadelphia, PA), R. Eisenberg (University of Pennsylvania), T. Minson (Cambridge University, Cambridge, U.K.), H. Browne (Cambridge University), and P. G. Spear (Northwestern University, Evanston, IL). We are indebted to Elisabetta Romagnoli for invaluable assistance. This work was supported by grants from the European Union (TargetHerpes), Fondo per gli Investimenti della Ricerca di Base autonomous and coordinated projects, Cofin-MIUR 40%, University of Bologna 60%, and Fondo Roberto and Cornelia Pallotti.

Abbreviations

Δ
deletion
HSV
herpes simplex virus
HVEM
herpesvirus entry mediator
pAb
polyclonal Ab
p.t.
posttransfection
WB
Western blot.

Footnotes

Conflict of interest statement: No conflicts declared.

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